The present invention relates to genes encoding proteins that regulate the pH of vacuoles, and the uses thereof.
In the flower industry, the development of novel or varied cultivars of flowering plants is important, and flower color is one of the most important traits of flowers. Although cultivars of various colors have been bred using conventional breeding by crossing, it is rare that a single plant species has cultivars of all colors. Thus, there is a need for the development of cultivars having a variety of colors.
The main components of flower color are a group of flavonoid compounds termed anthocyanins. It is known that a variety of anthocyanins occur in plants, and the structure of many of them have already been determined. The color of anthocyanins depends partly on their structures. Progress has been made in the study on the enzymes and genes involved in the biosynthesis of anthocyahins, and in some studies molecular biological techniques and gene introductions into plants were used to change the structure of anthocyanins, leading to changes in the color of flowers (Holton and Cornish, Plant Cell, 7:1071 (1995); Tanaka et al., Plant Cell Physiol. 39:1119 (1998)). The color of anthocyanins also depends on the pH of the aqueous solution, and the same anthocyanin may appear blue when the pH of the aqueous solution is neutral to weakly alkaline (Saito and Honda, Genda Kadaku (Chemistry Today), May 1998, pp. 25).
It is also known that since anthocyanins are present in the vacuole of the cell, the pH of vacuoles has a great impact on the color of flowers (Holton and Cornish, Plant Cell, 7 (1995); Mol et al., Trends Plant Sci. 3:212 (1998)). For example, in morning glory (Ipomea tricolor), it is known that the reason why red-purple buds bloom into blue flowers is that the pH of vacuoles in petal epithelium rises from 6.6 to 7.7 (Yoshida et al., Nature 373:291 (1995)).
It is thought that the vacuole of plant cells is regulated by vacuolar proton-transporting ATPase and vacuolar proton-transporting pyrophosphatase (Leigh et al., The Plant vacuole (1997), Academic Press), but the mechanism of how these proton pumps are involved in the color of flowers has not been elucidated. It was also known that a sodium ion-proton antiporter (hereinafter referred to as Na+xe2x80x94H+ antiporter) exits in plant vacuoles and that the Na+xe2x80x94H+ antiporter transports sodium ions into vacuoles, depending on the proton concentration gradient between the outside and the inside of vacuoles, whereupon protons are transported outside of vacuoles resulting a reduced proton concentration gradient.
Furthermore, the Na+xe2x80x94H+ antiporter is thought to be a protein with a molecular weight of about 170,000. However, there are many unknown factors involved in the regulation of pH of vacuoles, and the mechanism of regulating the pH of vacuoles, in particular the petal vacuoles, is uncertain (Leigh et al., The Plant Vacuole (1997), Academic Press). The pH of plant vacuoles has never been artificially raised, nor have any industrially useful traits been obtained, and its association with flower color is unknown.
It is known that the Na+xe2x80x94H+ antiporter gene, with a molecular weight of about 70,000, has been cloned from Arabidopsis, and a yeast into which this gene was introduced has acquired salt tolerance (Gaxiola et al., Proc. Natl. Acad. Sci. USA 96:1480-1485 (1999)), but it is not known how this antiporter regulates the pH of vacuoles in plant cells or how it is associated with flower color.
On the other hand, in petunias, seven loci are known to be involved in the pH regulation of petal vacuoles, and it has been proposed that the pH of petal vacuoles increases when one of them turns homozygously recessive (van Houwelingen et al., Plant J. 13:39 (1998); Mol et al., Trends Plant Sci. 3:212 (1998)). One of them, Ph6, has already been cloned and was found to be a kind of transcription regulating factor (Chuck et al., Plant Cell 5:371 (1993)), but the actual biochemical mechanism involved in the pH regulation of vacuoles is unknown.
In morning glory (Ipomea nil), the analysis of mutants revealed that a number of loci are associated with the color and shape of leaves and flowers and that 19 of them are highly mutable (Iida et al., Shokubutsu Saibo Kogaku Series (Plant Cell Engineering Series) 5 (1996) pp. 132, Shujunsha; Iida et al., Annal. New York Acad. Sci. (1999) pp. 870). Among them, the one locus defined by the recessive mutation that results in purple flowers instead of blue flowers is termed the Purple locus (T. Hagiwara, The genetics of flower colours in Phrarbitis nil. J. Coll. Agr. Imp. Univ. Tokyo 51:241-262 (1931); Y. Imai, Analysis of flower colour in Pharbitis nil. J. Genet. 24:203-224 (1931)), and one allele of mutable mutation that results in flowers that produce blue sectors in purple petals was termed purple-mutable (pr-m) (Imai, J. Coll. Agric. Imp. Univ. Tokyo 12:479 (1934)). The gene derived from the Purple locus is termed Purple gene.
The blue portion is believed to be derived from somatic reverse mutation from the recessive purple, and germ cell revertants can also be separated. An allele produced from the reverse mutation of these revertants are termed herein Purple-revertant (Pr-r). Such a classical method of genetic analysis had been performed on this Purple gene, but the identity of the Purple gene and its association etc. with the pH regulation of petal vacuoles were totally unknown.
It is believed that if the pH of vacuoles could be modified, for example if the pH of vacuoles could be raised, flower color could be turned blue. Representative plant species that lack blue colors include roses, chrysanthemums, carnations, gerberas and the like, which are very important cut flowers. Though the importance of modifying pH of vacuoles has been recognized, the identities of proteins that regulate the pH of petal vacuoles are unknown and therefore the isolation of genes encoding them has been in great demand.
The present invention provides a gene of a protein that regulates the pH of vacuoles in plant cells, preferably a gene of a protein that transports protons in vacuoles, more preferably a Na+xe2x80x94H+ antiporter gene. By introducing the gene of the present invention into a plant and allowing it to be expressed, flower color can be controlled and, preferably, can be turned blue.
Thus, the present invention provides a gene encoding a protein that regulates the pH of vacuoles. This gene is, preferably, a gene encoding a Na+xe2x80x94H+ antiporter, for example a gene encoding a protein that has the amino acid sequence as set forth in SEQ ID NO: 2, or a gene encoding a protein that has an amino acid sequence modified by the addition or deletion of one or a plurality of amino acids and/or substitution with other amino acids in the amino acid sequence as set forth in SEQ ID NO: 2 and that, has an activity of regulating the pH of vacuoles; a gene encoding a protein that has an amino acid sequence having a identity of 20% or more with the amino acid sequence as set forth in SEQ ID NO: 2 and that has an activity of regulating the pH of vacuoles; or, a gene that hybridizes to part or all of a nucleic acid having a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 2 under a stringent condition, and that encodes a protein having an activity of regulating the pH of vacuoles.
The present invention also provides a vector comprising the above gene.
The present invention also provides a host cell transformed with the above vector.
The present invention also provides a protein encoded by the above gene.
The present invention further provides a method of producing a protein that has an activity of regulating the pH of vacuoles, said method comprising culturing or growing the above host cell and then harvesting said protein from said host cell.
The present invention also provides a plant in which said gene or said vector has been introduced or a progeny thereof having the same property as said plant, or a tissue thereof.
The present invention also provides a cut flower of the above plant or a progeny thereof.
The present invention further provides a method of regulating the pH of vacuoles comprising introducing the above gene or the above vector into a plant or plant cells and then allowing it to be expressed.
The present invention further provides a method of controlling the flower color of plants comprising introducing the above gene or the above vector into a plant or plant cells and then allowing said gene to be expressed.